Summary: Although it is known at a molecular level that anthrax LT acts to cleave MAPKKs, its exact role in the overall pathogenesis of anthrax infection is unknown. There is a consensus that LT has at least one indisputable action: LT lyses macrophages at threshold concentrations. However, data regarding the in vivo action of LT on macrophages is lacking. Additionally, significant controversies remain unresolved regarding the in vitro effects of LT. For example, it has been reported that IL-1 and TNF-a produced by macrophages in response to LT are critical mediators underlying its lethal effects, while other investigators have reported that LT may inhibit rather than induce pro-inflammatory cytokine production by macrophages. Regardless of the underlying reason(s) for this discrepancy, it is clear that establishing where and when LT acts will be essential in predicting the efficacy of therapies that target this pathway. In this regard, it has not been established whether cytokine or anti-cytokine therapies can attenuate the action of LT in vivo or whether these potential therapies could augment host responses to infection. With the scientific tools that are currently available (e.g., gene-targeted mice), we can now establish definitively whether TNF-a and IL-1 pro-inflammatory cytokine pathways are required for LT action in vitro and in vivo. Elucidation of the involvement of other cytokine pathways is also warranted. For example, while promoting cellular immunity, the IL-12/Stat4 signal transduction pathway down-regulates other immune responses, including the deleterious responses to endotoxin in mouse models. Therefore, it is conceivable that modulation of Stat4 signaling could decrease the systemic shock response to various bacterial toxins, including anthrax LT. Additionally, it is unknown whether cytokines that down-regulate certain myeloid APC functions (e.g., IL-4 and IL-10) or those that promote cell survival (e.g., M-CSF) can attenuate the response of macrophages to LT. Moreover, it is unknown whether concomitant exposure of macrophages to anthrax edema toxin (ET), which would be expected in natural infections, affects LT actions. To address these questions, we have obtained E. coli expression vectors for the three anthrax toxin factors (lethal factor, edema factor, and protective antigen) from Dr. John Collier. In collaboration with Dr. Kathleen Clouse and Dr. Steven Kozlowski in DMA, who has experience in the area of protein production in E. coli, we intend to produce toxin components in large-scale quantities with high purity. It will be essential for us to produce endotoxin-free components. Through the use of blocking antibodies and gene-targeted mice, we will determine the role of the above-mentioned cytokine pathways in LT action. Also, we will investigate whether anthrax ET has effects on LT action in vitro and in vivo. Finally, we will ascertain whether toxin action is attenuated by inhibitory cytokines. Ultimately, one of the goals of our studies will be to determine the effect of cytokine and anti-cytokine therapies when the toxins are used in vivo. If we can prove that certain cytokines play a role in toxin activity, we will next investigate whether existing cytokine or anti-cytokine therapies are efficacious in attenuating responses to anthrax LT. Additionally, we plan to confirm these findings using live infection models in collaboration with outside investigators who have access to adequate containment facilities (currently unavailable at CBER).